Biological medicament



Patented Feb.- 12, 1935 g UNITED STATES PATENT OFFEQE BIOLOGICALMEDICAMENT Newell S. Ferry, Detroit, Mich, assignor to Parke,

Davis & Company, Detroit, Mich, a corporation of Michigan No Drawing.Application October 13, 1930, 7 Serial No. 488,547 7 '7 Claims. (Cl.167'78) The invention relates to the production of for the first time ingrowing the organism (Mic. biological medicaments for the treatment ofgonorrhece) luxuriantly with a heavy pellicle in gonorrheal infectionsand more particularly to a liquid culture medium. the production of atoxin and an antitoxin The toxin is obtained by inoculating asuitspecific to the organism causing the disease. able culture mediumwith a pure culture of the 5 A known organism, Mic. gonorrhece, isresponorganism (Micrococcus gonorrhece, Diplococcus sible for producinggonorrheal infections and a gonorrheae) specific to gonorrhea andgonorrheal variety of bacterial preparations derived from infections.The culture medium must be a liquid this organism have been proposed forthe treatmedium in which the organism grows readily,

10 ment of the disease although without decisive such as glucose brothwith ascites fiuid or horresults such as characterize the treatment ofmone broth with or Without ascites fiuid. After diphtheria or scarletfever. Such products have incubation for two to six or more days, thebroth been antigonococcic serum, gonococcus bacterins, cultures: aresterilized preferably, though not a non-toxic gonococcus antivirus, etc.necessarily, by passage through germ proof fil- The view is at presentaccepted by workers in ters. After the culture is sterilized it is best15 this field that Mic. gonorrhece does not produce kept at refrigeratortemperature. The toxin is an extracellular toxin and hence thepreparathen standardized preferably by the-skin test tion of a suitabletherapeutic agent based upon method. the production of a true toxin anda true anti- In preparing gonococcus toxin in liquid media.

toxin has not been considered as hopeful. it is preferable to developand maintain the orw I have discovered that Mic. gonorrhece when ganismsin the form of a pellicle or film on the grown under suitable conditionsproduces a heresurface of the media. The method which I have toforeunrecognized true extracellular toxin and found ,to give the mostsatisfactory results is to I have invented methods of securing thistoxin grow the organisms first in test tubes of hormone 5 in highconcentration and of producing from it broth or some enriched media forseveral days -5 a true antitoxin which can be used for quantitaor untila well developed film or pellicle has tive neutralization of the toxin.formed. As much of this pellicle as possible is As a result of thisinvention new standardizedv then transferred by means of a wire loopfrom medicaments can be made available for the treatthe test tube to thefinal culture flask and is so ment of gonorrheal infections, namely ananticarefully floated on the surface of the media in toxin, a toxin, andcombinations of toxin and the flask instead of being planted orinoculated antitoxin. 7 into the media as is customary with mostbacteria.

The toxin may be used for'determining suscep- During the process ofincubation of the culture tibility to gonorrheal infections, forproducing flasks care is exercised to insure thatthe flasks 5 theantitoxin and for standardizing the latter. are not moved orjarred. Ifdislodged, the pellicle Possibly it may also be of value in thetreatwill rapidly sink to the bottom of the flask, ment of the conditioneither by injection as either delaying or entirely prohibiting theforsuch or in its neutralized form. The artificially mation of the toxindue to the death of many made antitoxin is useful for the directtreatment of the organisms.

40 of the infection and especially for the relief from The toxic natureof this new gonococcus toxin 40 toxic symptoms. It may also be used forthe is shown by the fact that when injected into preparation ofneutralized toxin as well as for mice it produces fatal results and wheninjected the identification of Mic. gonorrhea: because of intradermallyinto rabbits, it develops inflamits specific toxin neutralizingproperties. matory lesions in the skin where injected. Also wheninjected subcutaneously and intradermal- 45.

v 7 ly in high dilutions into the human, it produces As set forth above,no one has heretofore oban irritation which causes inflammatory lesionstained a toxin specific to Mic. gonorrheoe but at the site of injection.by reason of my invention such toxin has now been obtained and it hasbeen produced under such conditions that it is now possible to make Tostandardize the toxin, the undiluted gonuse of the same in themanufacture of biologiococcus toxin is diluted and compared with a calmedicaments for the treatment of gonorrheal diluted gonococcus toxinwhich has already been infections. standardized by a skin reaction orother spe- 45 Preparation of gonococcus toxin Standardization ofgonococcus tomin In carrying out my invention I have succeeded cificreactions on susceptible individuals or ani-' 55 or animal.

male. In carrying out the standardization test or the comparison betweenthe new toxin and the standard toxin, various diluations are made ofthe'new lot of toxin and these are compared Dilute or'weak solutions ofthe toxin oLk'nown strength may be employed to determinesusceptibilityto gonococcus tdxinend Mic. gonorrhea: f

by means of. the'skin test. "The gonococcus toxin of known strength 'ispreferably diluted with physiologic salt solution so that 0.1 cc. willrep- The test consists of an intracutaneous injection of 0.1 cc. of--the diluted standardized toxin preferably on the flexor resent one skintest dose.

' surface of the forearm. The reaction is noted at 7 serum reactions.

the end of 24 hours. The reaction'is characterized by an area ofreddening which should be at least 1 cmjin diameter. This reaction iscontrolled by injecting the same amount of toxin,

in corresponding position on the other arm, the toxicityof which hasbeen destroyed by heating or by neutralizing with gonococcus antitoxin.1

. Preparation of gonococcus antitoxin The gonococcus toxin is used forthe production of the gonococcus antitoxin. Animals immunized withinjections of gonococous toxin will produce, an antitoxinv which willneutralizegonococcus toxin when mixed. with it in proper quantities.

The preparation of the gonococcus antitoxin is preferably carried outinthe following manner: The gonococcus toxin prepared in the manner abovedescribed is. injected into animals, preferably horses, in graduallyincreasing doses, beginning with a dose small enough not to producesevere symptoms in the animal, and gradually increasing. to .thetolerance of the animal. The toxin used in these injections need notnecessarily be sterile for in some cases it may be advisable tointroduce, the living organism with the toxin. When a sterile toxin isinjected it may either be a product in which the organisms have beenkilled and are still contained in the liquid, or

it may be a solutionzwhioh has been freed from all organisms. Afterpreliminary tests show the antitoxin is of sufficient strength as testedagainst the gonococcus toxin, the horse or other animal is bled and theserumor plasma may be concentrated and refined in order to avoidunnecessary The gonococcus antitoxin. is then standardized, bydetermining how many skin test doses of a' standard gonococcus toxin onecubic centimeter of the antitoxin will neutralize.

The standardization of the gonococcus toxin and the production,refinement, and standardizationof the antitoxin, make the use of theseproducts practical as a routine measure in the identification of theMic. gonorrhea; and the r treatment of gonorrheal infections.

The indentification of Mic. gonorrhea? The use of the toxin serves as amethod of recognizing the organism which is capable of producinggonorrheal infections. The organism to be identified is isolated in pureculture and grown on suitable media, preferably richer than ordinarybeef broth. After, a reasonableincubation the culture is clarified oforganisms or sterilized by filtration, centrifugation or other suitablemeans and the sterile filtrate is tested for its toxicity, firstcomparing it with agonococcus toxin of known strength. After it isdetermined that it'produces a skinreaction on individuals known to besusceptible to gonorrhea, it is mixed with the new' gonococcus antitoxinwhich is now shown to neutralizethe newly discovered gonococcus toxin.If the toxin is so neutralized by the gonococcus antitoxin that it willno longer produce a skin reaction, it may be considered that theorganism under consideration is capable of producing a. toxin specificto gonorrheal infections and that the organism is also specific.

cal use, particularly for the preparation of the antitoxin. In theappended claims the term toxin is intended to cover any of the abovemen- It is further to be understood that gonococous I tioned forms whilethe term sterile-toxin refers to a product which may or may not containkilled organisms but contains no living organisms. Whenever it isintended'to refer to the toxin it: self free from all organisms, I havespecifically stated that the product is free from organisms.

What I claim as my invention is: l 1; A true extracellular toxinspecific to gonorrheal infections obtained from a culture of Mic.

gonorrheaeand capable: of being neutralized by gonococcus antitoxin. 2'.A true antitoxin specific to gonorrhealinfections comprising the serumobtained from an animal after the injection with. a true extracellulartoxin specific to gonorrheal infections andwhich serum is capable ofneutralizing said true extracellular toxin. 7 I 3. A true extracellulartoxin specific to, gonor rheal infections obtainedfrom a culture ofMi'c.- gonorrheoe and capable of being neutralized by gonococcusantitoxin, said toxin being sterile and free from bacterial cells.

4. A true antitoxin specific to gonorrheal infec-' tions comprising aserum-obtained fromian animal after injection with a true-extracellularsterile" bacteria-free toxin specific to=gonorrheal infections whichserum is capable of neutralizing said extracellular toxin. v

'5. The process of obtaining a true: extracellular gonococcus toxincomprising growing Mic. gonor= rhew on a suitable liquid culture mediumto form a pellicle, maintaining said medium at incubating temperaturesuntilsaid pellicle begins to disin-. tegrate due to the'natural death ofthe organisms and eliminating said organisms, before'any sub-J stantialautolysis takes'place. Q a y 6. The'process of obtaininga trueextracellular gonococcus toxin'comprising growing Mic. gonorrhew on asuitable liquid culture medium-to form.

a pellicle, maintaining said medium I under incubating conditions for aperiod not substantially from the liquid and sterilizing the liquid.

exceeding six days, separating the organisms- 7. The process ofobtaining an antitoxin specific to-gonorrhea infections comprisinggrowing Mic. gonorrhea; on a suitable liquid culture medium for a periodof time not substantially greater than day's, separating the organismsfromsaidliquid thereby forming a true'extracellular toxin, in.--'jectingsaid true extracellular toxin into an an:-. imal and obtainingfrom the blood serum of said animal a true antitoxin specific to'gonorrheaim. fections; 1

. NEWELL S

